PREGUNTA DE INVESTIGACIÓN

In chapter 5.1, we studied the internalization of EGF receptor-targeted polyplexes with 2 kDa PEG into HuH7 cancer cells under static conditions. We demonstrated that the full-length EGF ligand modifies the uptake kinetics of polyplexes, promoting fast receptor-dependent polyplex internalization, whereas untargeted polyplexes exhibit slow uptake. The positive effect of ligand installation on the transfection efficiency of EGF-PEG2-polyplexes was confirmed by other in vitro and in vivo studies. To analyze if the EGF ligand promotes the adhesion of polyplexes to EGFR expressing cancer cells, we compared the binding of EGF-equipped an untargeted polyplexes in our microfluidic flow chamber. Experiments were performed analogue to the previously described flow experiments. Figure 5.18 depicts the adhesion kinetics of EGF-PEG2 and PEG2 polyplexes within 30 minutes under flow conditions (flow rate = 2 ml/minutes; shear stress = 2.6 dyn/cm2). Unexpectedly, similar normalized particle numbers were detected in the first 20 minutes of incubation. Within 20 to 30 minutes of incubation only a slight increase in particle adhesion could be observed for the EGF polyplexes.

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Additional experiments were performed after 1.5 hours of incubation, also here only a low effect of the EGF ligand within one magnitude was observed (data not shown). These results suggest that the cellular adhesion of PEG2-polyplexes is dominated by non-specific interactions and not by receptor- ligand interactions.

Figure 5.18 Cellular attachment of EGFR-targeted and untargeted polyplexes under flow. HuH7 cells in a microfluidic channel were subjected to EGF-PEG2 polyplexes (magenta squares) or untargeted PEG2 polyplexes (blue circles) for 30 minutes under flow conditions (2ml/min flow rate). The kinetics of particle attachment was followed by widefield-fluorescence microscopy. Each data point represents the normalized number of polyplexes bound to a single cell. Data were approximated by linear fitting.

5.3.4 Discussion

In this study a microfluidic device was successfully established that allows the detection of nanoparticle interactions with cells and physiological biomolecules at different shear rates. In a first set of flow-experiments we evaluated the adhesion of untargeted polyplexes with different PEG shielding to collagen and to HuH7 cancer cells. We revealed that polyplexes with 2 kDa PEG show strong adhesion to collagen. In vivo this interaction may lead to strong retention of polyplexes in the extracellular matrix. The polyplex binding to collagen could be reduced by elongation of the PEG linker (20 kDa PEG). Polyplexes with 20 kDa PEG also exhibited reduced non-specific binding to HuH7 cancer cells compared to polyplexes with 2kDa PEG. In a second set of experiments the adhesion of EGFR-targeted polyplexes and untargeted polyplexes with 2 kDa PEG to EGFR overexpressing HuH7 cancer cells was compared. Unexpectedly the EGF ligand had only low effect on the binding of polyplexes to HuH7 cells; the unspecific electrostatic interactions of the positively charged polyplex core seemed to dominate the adhesion process. Consequently, as a next step it would be of interest to monitor cell adhesion of targeted particles with

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reduced surface charge under flow conditions. One possibility would be to use targeted polyplexes with long 20 kDa PEG linkers for these experiments. However, we learned from our first microfluidic experiments that calibration of the experiments with self-assembly polyplexes is very challenging. The number and size of the polyplexes varies for each polyplex batch and aggregation of polyplexes can be induced over time in the cell medium. Implementation of longer PEG molecules or targeting ligands can affect the self-assembly process resulting in altered polyplex structure, DNA packaging and surface charge. The attachment of particles to cells under flow however is affected by each of these variables. The particle size and density determines the diffusion and sedimentation velocity of particles. The probability of cell surface binding increases with the number of deposited particles and additionally depends on various physico- chemical parameters such as hydrophobicity and surface charge. We tested different external and internal controls in our experiments to estimate the size and number of our particles in solution and particle deposition to the cells in the microfluidic channel, such as spin-coating of particles to a coverslip, particle sedimentation onto collagen under static conditions, fluorescence correlation spectroscopy (FCS) measurements in solution, and simultaneous multi-channel experiments with different cell-types and surface coatings. Nevertheless, exact quantification remained difficult. Therefore we decided to use commercially available model beads for future targeting experiments that should provide homogeneous size and defined composition. First experiments with PEGylated latex beads revealed a promising effect of installation of a B6 ligand on the specific binding to transferrin receptor overexpressing cancer cells. Another strategy would be to use stabilized polyplexes with internal cross-links for future experiments that should remain functional over longer time periods. Despite some challenges, the microfluidic setup has great potential to elucidate interesting questions in the future. For example multiple microfluidic channels can be simultaneously flushed with a particle solution to screen the interactions of newly developed materials with selected molecules and cells in parallel. We are currently setting up a four-channel system in which the binding of particles to target cancer cells, non-target endothelial cells, extracellular matrix components and blood proteins can be evaluated at the same time. Furthermore, by combining highly-sensitive fluorescence imaging with particle tracking, the mechanisms of particle binding to targeting receptors, the glycocalyx or specific surface molecules can be enlightened. Next to adhesion studies, information on the effect of shear stress on the internalization kinetics of nanoparticles can be gained. Also the effect of residual molecules from the fabrication process of particles (e.g. free polymer, ions or surfactants) on particle binding, uptake and cell viability can be evaluated.

Furthermore, in the microfluidic flow chamber the effect of nanoparticles on circulating non-adherent metastatic cells or immune cells can be analyzed as well. Also, new types of gene or drug nanocarriers for the therapy of multiresistant bacteria cells in the blood flow, or for the therapy of cardiovascular diseases may be examined.

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6 Improved scaffolds for gene and drug delivery

A plethora of polymer and dendrimer scaffolds with multiple modifications are available for the delivery of drugs and nucleic acids to cells and laboratories around the globe are working steadily on the design of improved molecules with high loading capacity, reduced aggregation, specific functionalities and low toxicity. When searching for publications dealing with “polymer and gene” in the U.S. library of medicine of the National Institutes of Health, almost 67.000 entries are displayed (status, May 2013). The production process of these scaffolds is often challenging due to complex, multi-step synthesis and purification. Furthermore lots of the produced scaffolds are polydisperse mixtures of molecules with a broad range of different sizes and conjugation sites. Here we analyze two novel nanocarrier scaffolds with improved production process for their ability of gene and drug delivery.

In the first to part of this chapter we apply live-cell imaging to compare gene delivery by a new 4-arm- PEG dendrimer hybrid and a 2-arm PEG dendrimer hybrid that can be synthetized in only four steps with simple purification methods (section 6.1). The constructs were synthetized and characterized in the group of Prof. Craig Hawker at UC Santa Barbara and results described in this chapter are taken from our joint publication in the journal Biomacromolecules9. In section 6.2 we study a similar internally functionalized dendrimer to which coumarin was attached as a model delivery agent via cleavable ester bonds337. The effect of coumarin loading to the dendrimer and intracellular coumarin delivery was monitored by confocal microcopy. Experiments with coumarin-loaded dendrimers were performed in collaboration with Dr. Roey Amir (UC Santa Barbara, since 2012 University of Tel Aviv) and Dr. Lorenzo Albertazzi (UC Santa Barbara, since 2012 University of Eindhoven) during the research visit of Lorenzo Albertazzi at our department. In section 6.3 cellular interactions of a novel sequence-defined polymer with PEG shielding and EGF conjugation for EGFR targeting were monitored. The synthesis of this polymer with defined architecture and conjugation sites is based on the solid phase assisted coupling of artificial amino acid subunits published recently by Schaffert et al.338 Synthesis and characterization of the EGFR-targeted polyplexes was performed by Ulrich Lächelt, reporter gene expression assays were provided by Petra Kos (both from the group of Prof. Ernst Wagner, pharmacy department, LMU Munich).

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